DESCRIPTION: The long term goal of the proposed work is to understand the molecular biological and biophysical processes that are responsible for DNA-damage induced mutagenesis in Escherichia coli. This proposal will exploit the analytical power of experiments with vectors that carry a specifically located and defined abasic site or UV photoproduct. These experiments, unlike those using mutagen treated cells, can provide independent estimates of the frequency of translesion replication, the error frequency of translesion replication, and the types of mutations induced. The first group of specific aims uses the vectors as tools to investigate the in vivo functions of proteins that promote mutagenesis, and to explore genetic events associated with translesion synthesis. The mutagenic properties of different lesions will be determined in strains deleted for genes encoding subunits of DNA polymerase III and expressing either the UmuD'C, MucA'B, or RumA'B complex, with the aim of identifying the subunits with which these mutagenesis proteins interact, and the investigating the reasons for their altered characteristics. The possible role of DNA polymerase II in the bypasss of abasic sites will be examined. The vectors will also be used to examine template switching, and the properties of lesions during leading and lagging strand synthesis. The second group of specific aims is directed towards understanding the characteristics of particular mutagenic lesions, and the influence on them of flanking nucleotides. The properties of the T-C and C-C cyclobutane dimers will be explored, and the deamination rates of cytosine measured in vivo. Nuclear magnetic resonance spectroscopy will be used to examine the structure of oligomer duplexes containing different isomers of the T-T and T-C(6-4) adducts, to investigate the mechanisms responsible for selective insertion of guanine opposite the pyrimidinone. This work will lead to a better understanding of mutagenesis induced by UV, a serious environmental mutagen, and mutagenic processes in general, which are implicated in cancer induction.